Search

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli , resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli . In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Izghirean, Waidacher, Kittinger, Chyba, Koraimann, Pertschy and Zarfel.)

A collection of 611 Pseudomonas isolated from 14 sampling sites along the Danube River were identified previously by MALDI-TOF MS with the VITEK MS system and were grouped in 53 clusters by their main protein profiles. The strains were identified in the present study at the phylospecies level by rpoD gene sequencing. Partial sequences of the rpoD gene of 190 isolates representatives of all clusters were analyzed. Strains in the same MALDI-TOF cluster were grouped in the same phylospecies when they shared a minimum 95% similarity in their rpoD sequences. The sequenced strains were assigned to 34 known species (108 strains) and to 32 possible new species (82 strains). The 611 strains were identified at the phylospecies level combining both methods. Most strains were assigned to phylospecies in the Pseudomonas putida phylogenetic group of species. Special attention was given to 14 multidrug resistant strains that could not be assigned to any known Pseudomonas species and were considered environmental reservoir of antibiotic resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50% of the Pseudomonas species in the Danube River able to grow in the isolation conditions have been identified at the species level. Main objectives were the confirmation of the correlation between the protein profile clusters detected by MALDI-TOF MS and the phylogeny of Pseudomonas strains based on the rpoD gene sequence, the assessment of the higher species discriminative power of the rpoD gene sequence, as well as the estimation of the high diversity of Pseudomonas ssp. along the Danube river. This study highlights the Pseudomonas species diversity in freshwater ecosystems and the usefulness of the combination of MALDI-TOF mass spectrometry for the dereplication of large sets of strains and the rpoD gene sequences for rapid and accurate identifications at the species level., (Copyright © 2020 Mulet, Montaner, Román, Gomila, Kittinger, Zarfel, Lalucat and García-Valdés.)

Spread and persistence of antibiotic resistance pose a severe threat to human health, yet there is still lack of knowledge about reservoirs of antibiotic resistant bacteria in the environment. We took the opportunity of the Joint Danube Survey 3 (JDS3), the world's biggest river research expedition of its kind in 2013, to analyse samples originating from different sampling points along the whole length of the river. Due to its high clinical relevance, we concentrated on the characterization of Pseudomonas spp. and evaluated the resistance profiles of Pseudomonas spp. which were isolated from eight sampling points. In total, 520 Pseudomonas isolates were found, 344 (66.0%) isolates were identified as Pseudomonas putida, and 141 (27.1%) as Pseudomonas fluorescens, all other Pseudomonas species were represented by less than five isolates, among those two P. aeruginosa isolates. Thirty seven percent (37%) of all isolated Pseudomonas species showed resistance to at least one out of 10 tested antibiotics. The most common resistance was against meropenem (30.4%/158 isolates) piperacillin/tazobactam (10.6%/55 isolates) and ceftazidime (4.2%/22 isolates). 16 isolates (3.1%/16 isolates) were multi-resistant. For each tested antibiotic at least one resistant isolate could be detected. Sampling points from the upper stretch of the River Danube showed more resistant isolates than downriver. Our results suggest that antibiotic resistance can be acquired by and persists even in Pseudomonas species that are normally not in direct contact with humans. A possible scenario is that these bacteria provide a reservoir of antibiotic resistance genes that can spread to related human pathogens by horizontal gene transfer.